Retrovirus detection

ABSTRACT

A method, and groups of cells and retroviruses useful in the performance of the method, of testing for the presence or amount of a first retrovirus in a specimen. A replication defective retroviral construct having a marker sequence is exposed to the specimen and the presence or amount of a recombinant retrovirus carrying the marker sequence is tested for. The replication defective vector may be disposed in cells, with the specimen being tested for the presence or amount of a retrovirus, or it may be disposed in a retrovirus, with the specimen being a group of cells which are tested for the presence or amount of said first retrovirus. In either case, where a first retrovirus complements the defective vector, a recombinant retrovirus carrying the marker sequence will be produced. The first retrovirus to be tested for may be of a previously known, or unknown type, and may be any of a group of retroviruses.

This application is a continuation of application Ser. No. 08/016,664,filed on Feb. 10, 1993,now abandoned which was a file wrappercontinuation application of Ser. No. 07/373,458, filed on Jun. 30, 1989now abandoned.

FIELD OF THE INVENTION

This invention relates to retroviruses. It further relates to a methodof testing for the presence of a retrovirus in a specimen, typically apreviously unknown retrovirus, and to techniques of testing forrheumatoid arthritis based upon the presence of a retrovirus.

BACKGROUND

Retroviruses are single-stranded RNA viruses. Upon infection of a cellby a retrovirus the retroviral RNA genome is transcribed into itscorresponding double-stranded DNA by a reverse transcriptase enzymewhich is coded for by the viral genome. This DNA then enters the nucleusand integrates into the host DNA using an integrase enzyme which is alsocoded for by the viral genome. The integrated viral DNA ("proviral" DNA)becomes a component of the host genome, replicating with it and beingpassed on to daughter cells in a stable manner. The proviral DNA is alsoin general transcribed to give viral RNA molecules which code for themajor viral proteins, namely the gag, pol and env proteins (the"packaging proteins"). The full length RNA transcript can be packaged bythe viral proteins into a viral particle which then buds off in a pieceof cell membrane, in which are embedded env-derived peptides. Thismembrane-coated viral particle is a fully competent viral particle andcan go on to infect other cells.

The genome of a retrovirus (in either the RNA or DNA form) can bedivided conceptually into two parts. The first, or "trans-acting,"category consists of the regions coding for viral proteins. Theseinclude the group specific antigen ("gag") gene for synthesis of thecore coat proteins, the "pol" gene for the synthesis of various enzymes(such as reverse transcriptase), and the envelope ("env") gene for thesynthesis of envelope glycoproteins. Other proteins may also be producedin different retroviruses from messages produced by various internalsplicing reactions. These viral functions determine a considerable partof the host specificity of a virus. In the murine leukemia virus ("MLV")family, for example, the env gene products interact with cell surfacereceptors and determine whether the virus is ecotropic (infects onlymice and rats), xenotropic (infects non-mouse species only), oramphotropic (infects mouse and other species, including human), and ithas been suggested that the host range of a virus can be altered byreplacing its env protein (see Milewski, Recombinant DNA TechnicalBulletin, Volume 9, Number 2, page 88 (1986)). The gag gene productsdefine specificity in mice with respect to two main types: N (NIHderived mice) or B (Balb/C derived mice).

In general, the second part of the retroviral genome is referred to asthe "cis-acting" portion and consists of the regions which must be onthe genome to allow its packaging and replication. This includes thepackaging signal on an RNA molecule, such as the viral RNA, whichidentifies that RNA molecule to viral proteins as one to beencapsidated, Long Terminal Repeats ("LTRs") with promoters andpolyadenylation sites, and two start sites for DNA replication. Thepromoters, enhancers, and other regions of the LTRs are also capable ofconferring tissue specificity such that the virus will only be"expressed" (i.e., transcribed and translated) in specific cell typeseven though it may infect others.

It has been recognized that the cis-acting elements are grouped ateither end of the viral genome, in or near the LTRs. Thus, the internalor "trans-acting" part of a cloned provirus might be replaced by a geneof choice to create a "vector construct" (see for example, F. Ledley,The Journal of Pediatrics, Vol. 110, No. 1, p. 1 (January, 1987)). Whenthe vector construct is placed in a cell where necessary viral proteinsare present, transcribed RNA should be packaged as viral particleswhich, in turn, will bud off from the cell. These particles will beindistinguishable in appearance from native virus particles, althoughthey carry only the RNA of the vector construct into a cell andintegrate it within that cell's genome. It is believed that the genewill then be functional in the new cell but, without the trans-actingpart of the viral genome, will be incapable of expressing those proteinsrequired for further virus production. Hence, the vector construct andthe virus carrying it are "replication defective" normally being unableto produce new viral particles in the cell. The vector construct can,however, be transcribed and express its gene product.

Retroviruses are known to be widely spread in non-human species and thecause of various pathogenic conditions. However, only a very smallnumber of retroviruses have been identified in humans, and these onlyrecently. In addition, new retroviral sequences have been detected incells of human origin (see Callahan et al., Science, Vol. 228, p. 1208;1985), although their significance remains unknown.

The cause of many human pathogenic conditions remains unknown. One suchpathogenic condition is human rheumatoid arthritis ("RA"). In variousstudies, viruses have been found to be present in body fluids from somehuman subjects suffering from RA, who were studied as part of a largergroup of RA subjects. However, the results in such studies have beeninconclusive in that, typically, a majority of the subjects did notexhibit the presence of the virus identified in the minority. Forexample, parvovirus has been demonstrated in RA synovial fluid in aminority of RA suffering patients. Annals of The Rheumatic Diseases,46:219-223 (1987). Epstein-Barr ("EB") virus infection also appears tobe present in a majority of patients with RA, but in no higherpercentage than in the normal population, although RA patients areapparently less able to regulate such infections than normal controlsubjects (Bardwick et al., Arthritis .Rheum., Vol. 23, p. 626, 1980). Aretrovirus has been identified by Brassfield et al., Arthritis Rheum,Vol. 25, p. 930 (1982), as the apparent cause of caprine arthritis, agoat arthritis clinically similar to human rheumatoid arthritis.

Previously, the standard techniques used in an effort to determine thepresence of an unknown retrovirus (e.g. one whose presence may besuspected but which has not previously been isolated or characterized)have been a reverse transcriptase assay, electron microscopy, andvarious immunologic and nucleic acid hybridization assays. For example,such techniques have been described by Weiss, R. (1982) in RNA TumorViruses Vol. I, pages 209-260 (Cold Spring Harbor Laboratory, N.Y.).

Complementation assays have been used to titer retroviruses that havealready been isolated and characterized. In other words, complementationassays are used to determine the ability of a known retrovirus to have aspecific biologic effect. For example, in the S+ L- assay a murineleukemia virus rescues a MSV replication-deficient virus, infects anuntransformed cell line and thereby induces a transformation event,Eckner and Kettrick, J. of Virology, Vol. 24, p. 383-90 (1979). Suchassays are time consuming, are highly dependent on the ability of theviruses to complement and the ability of the recipient cell to betransformed, and are subjective in view of the necessity to assess cellmorphology associated with the transformed state. In addition, theseassays deal with known, characterized retroviruses and are used simplyto titer those viruses.

As pointed out by Norval et al., Ann. Rheum. Dis., Vol. 38, p.507 (1979)and Hart et al., Ann. Rheum. Dis., Vol. 38, p. 514 (1979), the presenceof a retrovirus in a majority of patients with RA has not beendemonstrated, despite extensive efforts to determine the cause of RA.The failure to detect a retrovirus which is clearly associated with RA,or retroviruses associated with other human pathogenic conditions ofunknown etiology, could be the result of relative insensitivity of boththe reverse transcriptase assay and electron microscopy, and thelimitation of immunologic and nucleic acid hybridization assays whichonly search for virus proteins or nucleic acid sequences very closelyrelated to those known to exist. Further, many retroviruses can onlygrow in a few specific cell types, further exacerbating the difficultyin detecting previously unknown retroviruses. For example, it is knownthat human T-cell lymphotrophic viruses types I and II (HTLV I and HTLVII) can only be effectively grown in certain cells such as in T-cellgrowth factor-driven lymphocytes, or cell lines derived therefrom, asdescribed by Broder et al., Ann, Rev. Imunol., Vol. 3, p. 321 (1985).

Retroviral constructs (i.e. retroviruses carrying vector constructs)such as those described by Gruber et al., Science, Vol. 230, p.1057-1061 (1985), including pLPLM, have been developed as vehicles forgene replacement therapy. The plasmid pLPLM, a murine leukemiavirus-derived construct, has retroviral promoters (LTRs) and packagingsignals, but the pol, gag and env genes have been deleted and replacedby cDNA for human hypoxanthine phosphoribosyltransferase ("HPRT"). A. D.Miller et al., Proc. Natl. Acad. Sci. USA, ("PNAS") Vol. 80, p.4709-4713 (1983); J. K. Yee, Gene, Vol. 53, p. 97-104. It is known thatwhen viral constructs having the LTRs, packaging signal, and a gene ofinterest, are placed in a cell having a genome coding for a retroviruswith the packaging signal deleted (known as a "helper cell"), suchretrovirus will pseudotype the RNA transcribed from the vectorconstruct, which carries a packaging signal, into a recombinantretrovirus (a retrovirus carrying a replication defective retroviralvector, is sometimes referred to herein as a retroviral particle). See,e.g., PCT application No. WO 86/00922, published Feb. 13, 1986;Molecular Biology of Tumor Viruses, Second Edition, "RNA Tumor Viruses"Robert Weiss (Ed) Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., USA (1984); Mann et al., Cell, Vol. 33, p. 153-159 (May 1983);Watanabe et al., Molecular and Cellular Biology, Vol. 3, No. 12, p.2241-2249 (Dec., 1983); Watanabe et al., Proc. Natl. Acad. Sci., USA,Vol. 79, p. 5986-5990 (October, 1982); and Watanabe et al., EukaryoticViral Vectors, p. 115-121, editor Y. Gluzman, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., USA (1982). In addition,replication defective retroviral genes have been rescued from cells byinfecting the cells with a suitable retrovirus known to be present. Evenif the defective vector sequences are not appropriate to the rescuingviral proteins, such rescue happens at an appreciable frequency comparedto the frequency of a fully compatible defective vector (0.01%-1%). Forexample, see Linial, Journal of Virology, Vol. 38, No. 1, p. 380-382(April, 1981). Since a fully compatible defective vector will givesignals of about 10⁶ units/ml, frequencies described above should beeasily detectable.

SUMMARY OF THE INVENTION

The present invention provides a method of testing either laboratory orclinical specimens for the presence or amount of a retrovirus (sometimesreferred to herein as a "first" retrovirus). The specimen, or a portionthereof, is brought into contact with a retroviral vector which carriesa marker and which does not have the ability to replicate. The vector iscarried by either a retrovirus or a cell (i.e., the proviral form of thevector). The first retrovirus may be either a known or previouslyunknown retrovirus (an "unknown retrovirus" being one which has notpreviously been isolated and characterized). That is, in the case wherethe specimen is a sample to be tested for the presence of a retrovirus,the sample is not otherwise known to carry a retrovirus. In the casewhere the specimen is a group of cells, the cells are not otherwiseknown to be carrying the retrovirus. In the method described and claimedherein a first retrovirus, if present, complements the test reagentreplication defective retroviral vector so that a recombinant retrovirusmay be produced which carries the marker (the marker being selected sothat its presence can be conveniently determined). The replicationdefective retroviral vector used will be one with an encapsidation(i.e., packaging) signal, a marker sequence, and other requiredsequences (generally the 3' and 5' LTRs) so that when carried to a cell,and in the presence of the requisite enzymes, the defective vector canbe integrated into the cell's genome and express the marker. Thedefective vector may optionally additionally code for other proteins,including any of the gag, pol, or env proteins, required to produce acomplete retrovirus carrying the retroviral vector with the marker gene.However, it will be understood that the defective vector is "defective"in the sense that it is unable, in primary cells which may be used toreceive both the first and defective viral vectors, to produce completeviruses which can go on to infect secondary cells. Thus, the defectivevector will not code for all proteins necessary to produce furtherretroviruses therefrom.

In the practice of the invention, a test medium which contains primarycells or a retrovirus carrying the defective vector construct iscontacted with a specimen which is to be tested for a first retrovirussuch that the first retrovirus (if present in the specimen), maycomplement the replication defective retroviral vector and produce arecombinant retrovirus carrying the marker sequence. The production of arecombinant retrovirus carrying the marker is then tested for. It willbe understood that in the case where a specimen is to be tested for apreviously unknown type of retrovirus, it will not be known for certainwhether in fact that retrovirus will complement the replicationdefective vector at a detectable frequency. However, in the case where aspecimen is being tested for a particular type of known retrovirus, thenthe replication defective vector can be selected so that suchcomplementation is likely to be obtained.

In a preferred case, whether the first retrovirus is known or unknown,the defective vector can be designed to code for all packaging proteins,other than one. In this situation the chances of detecting the firstretrovirus may be increased, since the first retrovirus need onlyprovide a minimum complementation in the form of a single suitablepackaging protein which will complement the missing packaging protein.In an alternative arrangement, a plurality of groups of replicationdefective retroviral vectors can be exposed to the specimen or a portionthereof, the retroviral vectors of each group having a marker sequenceand a sequence which codes for a corresponding packaging proteindifferent from the packaging protein coded for by the sequences of theremaining groups, all of the groups together having sequences which codefor all packaging proteins required to produce a recombinant retroviruscarrying the marker sequence. Thus, portions of the specimen would betested against respective groups so that the members of the groupscannot complement one another. A single specimen portion could be testedagainst several groups which together carry all but one packagingprotein (so that the members of the group cannot completely complementone another). In these arrangements, the retrovirus being tested for maycomplement the retroviruses of some groups better than others, and thusoverall the chances of detecting the first retrovirus may be increased.This would be particularly true where the first retrovirus is of anunknown type.

In one preferred embodiment of the invention, primary cells which carrythe replication defective retroviral vector (in the proviral form) arecultured with the test specimen. Complementation of the defective vectormay be obtained directly or indirectly from a first retrovirus whichinfects the primary cells. Direct complementation results fromexpression by the first retrovirus, following its integration into theprimary cell genome, of the required protein (or proteins) for packagingand replication. Indirect complementation results from expression of therequired protein (or proteins) from the primary cell genome followingactivation by an exogenous agent (e.g., IL-1, IL-2 or other protein)which may be associated with the first retrovirus. It will beappreciated that the specimen can be patient cells to be tested for afirst retrovirus and that, in such case, for example, the defectiveretroviral construct can be carried as a vector into those patientcells.

Secondary cells may then be cultured with medium from the primary cells.If a first retrovirus was present in the specimen and the primary cellsare susceptible to infection by it, recombinant retroviruses (i.e.retroviral particles) may be produced in the primary cells and enter themedium, which recombinant retrovirus will carry the marker sequence. Ifthe secondary cells are susceptible to the recombinant retrovirus, theywill infect those cells and incorporate the marker sequence into thesecondary cells. The presence of the marker sequence in the secondarycells is tested for as an indication of the presence of a retrovirus inthe specimen.

In the case where the method is used to test for the presence of aparticular type of retrovirus in a specimen, primary cells can be chosenwhich are known to be susceptible to infection by that type ofretrovirus. Likewise, secondary cells would be chosen which are known tobe susceptible to infection by a recombinant retrovirus produced fromthe primary cells and in which the marker sequence will be expressed.

Various marker sequences can be chosen. However, the marker which willbe expressed in the secondary cells is preferably a sequence encoding aprotein selected from: (i) an enzyme; (ii) a protein which will enablegrowth of the secondary cells in a medium in which they would nototherwise grow; and (iii) an antigen and, in particular, one which willbe expressed on the cell surface. The presence of an expressible markersequence in the secondary cells is tested for by the presence or absenceof the marker sequence translation product. For example, the markersequence may code for HPRT and secondary cells will be selected whichare deficient in HPRT. Following culturing with media from the primarycell culture, the secondary cells will be examined for their ability togrow in hypoxanthine-aminopterin-thymidine (HAT) selection medium. Ifthe secondary cells grow in the HAT medium, this will be an indicationthat the specimen in fact contained a retrovirus. Of course, othermarker sequences can be used, e.g., the gene for β-galactosidase orluciferase.

Where a specimen is to be tested for an unknown retrovirus, or testedfor the presence of any of a group of known retroviruses, it can beuseful to choose the primary cells from a plurality of different celllines. As earlier described, a retrovirus can be highly specific as towhich cells they will infect and express their corresponding proteins.Thus, in the case of testing a specimen for an unknown retrovirus, usinga plurality of different cell lines increases the chances that aretrovirus, if present in the specimen, will productively infect theprimary cells. For the same reasons, it may also be desirable to selectsecondary cells which are from a plurality of cell lines.

It is further preferable that the secondary cells are not from any ofthe same cell lines as the primary cells in order that the secondarycells will be easily distinguished from the primary cells. Thisarrangement insures that, where the secondary cells are to be culturedwith primary cell medium, and in the event that some primary cells aretransferred into the secondary cell culture, they can be identified andremoved (such as by exposing the secondary cells to a medium which willselectively kill the primary cells). This prevents generation of a falsepositive test result if primary cells are accidentally transferred tothe secondary cell culture. Otherwise, the determination of the presenceof the marker sequence in the secondary cell culture would typically notdistinguish between primary and secondary cells expressing the markersequence.

In the case where the patient cells are used as primary cells, however,it is considered preferable to use as secondary cells the same cell typeas the primary cells since it will then be likely that any pseudotypedretrovirus produced from the primary cells will infect the secondarycells.

Generally, another technique which will increase the chances ofdetecting an unknown retrovirus in the specimen is to provide differentpackaging signals for the marker sequence. Thus, an unknown retrovirusin the specimen which may pseudotype a retroviral construct with onepackaging signal at a low efficiency, may pseudotype a retroviralconstruct with a different packaging signal at a higher efficiency.

In the case where primary cells are used which carry the replicationdefective retrovirus, a further technique to increase the chances ofdetecting a first retrovirus (particularly if of an unknown type), is toselect primary cells with their genome coding for at least one packagingprotein (i.e. gag, pol, env), preferably the genome of some of themcoding for a packaging protein different from that coded for by thegenome of others. For example, the genome of all of the primary cellscould code for a retroviral construct with a marker sequence, while thegenome of some of them also code for env protein, and still othersinstead also code for gag or pol protein. This arrangement will increasethe chance that recombinant retroviruses can be produced from theprimary cells upon exposure to a specimen with an unknown retrovirus,since the chances for complementation are increased. In a minimumcomplementation arrangement the genome of the primary cells has aretroviral construct which not only carries the marker sequence, but inaddition carries the sequences required to produce all of the proteins,except one, necessary to form a recombinant retrovirus carrying themarker sequence. In such a minimum complementation arrangement, thefirst retrovirus being tested for need only supply the missing proteinin the primary cells for production of recombinant retrovirus carryingthe marker sequence to occur. Thus, the chances of producing therecombinant retrovirus in the presence of a first retrovirus (to bedetected) may be increased.

A means of confirming the presence of a retrovirus where the retrovirustested for is of a previously known type, is to culture additionalprimary cells with a portion of the specimen and an antibody to theknown retrovirus and test for a reduced number of secondary cellscarrying the marker sequence as a result of the presence of theantibody.

Further steps which may be used with the above methods include a wash ofthe primary cells to remove inactive recombinant retrovirus prior toculturing the secondary cells with the primary cell medium. Preferablythe washing takes place about 1 day prior to this latter culturing step.This reduces the chance that any inactive recombinant retroviruses fromthe primary cells (which are potentially capable of binding withreceptors on the secondary cells, but are incapable of productivelyinfecting the secondary cells to express the marker sequence therein) donot interfere with productive infection of the secondary cells by liverecombinant retrovirus and reduce the sensitivity of the assay. Further,the primary cells or secondary cells, or both, can be treated to enhanceability of those cells to be infected with any recombinant retrovirus,e.g., with a detergent.

The present invention also provides cells which are useful in the abovedetection methods.

Using the above detection methods, the present inventors have found anassociation of a positive result with samples from patients sufferingfrom rheumatoid arthritis. This apparent retrovirus (or potentially agroup of retroviruses or a member of such a group) is referred tothroughout this application as "rheumatoid arthritis associatedretrovirus" (RA retrovirus), or equivalent expressions. As a result ofthis discovery, the present invention also provides for the testing ofrheumatoid arthritis in a human subject, which requires testing for thepresence of rheumatoid arthritis associated retrovirus in the subjectusing methods of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be described with reference to thedrawings, in which:

FIG. 1 illustrates a Moloney MLV genome with various deletions which canbe made thereto, the resulting genomes being useful in a minimumcomplementation assay described below (see Example 5, below);

FIG. 2 illustrates an amphotrophic envelope expression vector, useful toproduce an amphotrophic envelope protein for a recombinant retroviruscarrying a marker sequence (see Example 5, below).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Fluid specimens from a number of human subjects have been evaluated forthe presence of a retrovirus using the methods of the present invention.Primary cells from various cell lines were used, including an SV-40transformed fibroblast derived from a Lesch-Nyhan child (LNSV), whichwas transfected with pLS.increment.P.increment.LM (the resultingrecombinant cells being referred to as the LN.increment.P line) andselected for the HPRT-containing phenotype. pLS.increment.P.increment.LMis a plasmid carrying a murine leukemia virus-derived vector constructwith LTRs and packaging signals, but without pol, gag, and env genes,which were removed and replaced by the cDNA for a mutant HPRT.pLS.increment.P.increment.LM is the same as pLPL2 (Miller & Buttimore,Mol. Cell Biol., Vol. 6, p. 2895-2902 (1986)), but with the HPRT genemutated as described by Yee et al., Gene, Vol. 53, p. 97-104 (1987).Another primary cell line used was an EB virus transformed lymphoblastline derived from a Lesch-Nyhan child (cell line "1547"), which cellswere infected with a replication incompetent HPRT-containing retrovirus("LPL") and selected for the HPRT-containing phenotype. The preparationof such cells is described by Willis et al., J. Biol. Chem., Vol. 259,p. 7842-7849 (1984). The foregoing cell line does not spontaneouslyproduce HPRT-containing retroviruses. A third cell line used as aprimary cell source consisted of normal human cultured bone marrow,infected with a high titer preparation of a retrovirus containing thepLPLM vector construct from the producer line 7A2, as described inGruber et al., supra Science (1985).

Secondary cells should be selected which are likely to be susceptible toinfection by a recombinant retrovirus which may be produced from theprimary cells. The secondary cell should preferably be infectable by thepseudotyping virus. In the trials below, cell lines used as secondarycells in the assays were the HPRT-deficient rat 208F or murine B77 cells(with regard to these cell lines see, respectively, Miller et al., PNAS,Vol. 80, p. 4709-4713 (1983) and Miller et al., Molec. Cell. Biol., Vol.5, p. 431-437, (1985)). Other cells used as secondary cells includedhuman HPRT-deficient lymphoblasts (1547), fibroblasts (LNSV), orcultured human bone marrow cells. In the last case, the firstpseudotyped retrovirus can be re-pseudotyped by superinfection of thesecondary cells with murine amphotropic retrovirus (MA) (Miller et al.,supra. Mol. Cell. Biol., (1985)) and passage of the supernatant to 208For B77 cell lines (which act as tertiary cells). The advantage of thisprocedure is that the final progeny retroviruses are known to infect thefinal indicator cells.

The procedure followed in testing the specimens was generally asfollows. Primary cells containing the pLS.increment.P.increment.LM orvector construct were cultured with the specimen to be tested. After 3to 5 days, supernatant from the foregoing culture was placed onto anHPRT-deficient secondary cell line and after 24 hours the cell line wasgrown in HAT selection medium. During storage prior to use, thesecondary cell line is preselected in 6-thioguanine every four weeks toremove any HPRT-containing revertants. The primary tester line ismaintained as a precautionary measure in HAT till shortly before use toensure retention of the HPRT vector in the cell. When supernatants fromLN.increment.P are tested on secondary cell lines such as 208F or LNSV,a further negative control (208F or LNSV alone) is always performed tocheck for HPRT+ revertants. The cultivation of the LN.increment.P cellsfor 3 days after adding PBLs before transferring the supernatant tosecondary tester lines is a minimum time, but longer cultivation (up to30 days) also allows detection on the secondary line of transfer of HPRTvector, sometime with a larger signal. Thus, when setting up the assayit is optimal to utilize a time course, e.g,, 3, 7, 11, 14, 21, 30 days.

Control experiments were performed simultaneously with the precedingsteps, wherein a medium from untreated primary cells was passaged ontothe secondary cells to assure that the primary cells are notcontaminating the secondary cells and that the primary cells are notspontaneously producing retroviruses carrying the marker sequence. Thisprocedure also ensures that the secondary cell line has not developedHPRT-containing revertants. Positive controls were also run, whichrequired exposing the primary cells to MA, followed by the passage ofsupernatant therefrom to the secondary cells to assure that the assaycan detect retroviruses.

The secondary cell line was cultured for two weeks in HAT medium, theplates were washed, fixed in methanol, and the colonies counted. Eachcolony represented one HPRT-containing progeny retrovirus. The titer ofretroviruses in the specimen was determined by performing limitingdilution assays with portions of the specimen.

In addition to the above procedure, further and improved sensitivity wasachieved using the MA positive control as follows. Polybrene detergentwas added to the 208F cell line before infection with the supernatant.The use of this detergent tended to facilitate membrane adherence and,therefore, increase the ease with which retroviruses infected theprimary cells. The 208F cells were set up in microtiter wells a daybefore they were cultured with the specimen. The LNSV cell line, whichwas co-cultured with the clinical specimen, was washed the day beforesupernatant was transferred to the 208F secondary cells. This washingstep was utilized to remove previously made, but now inactive,interfering recombinant retroviral particles. If not removed, suchretroviral particles can potentially bind to, and block, receptors onthe secondary cells from productive infection by live recombinantretroviruses carrying the marker sequence.

EXAMPLE 1

On day one, 2×10⁵ cells from the line LN.increment.P were plated in DMEMplus 10% Fetal Bovine Serum on 60 mm dishes. This is the primary testline. The cells were grown in 10% CO₂ at 37°. On day four, polybrene wasadded to the media to 4 μg/ml at least 2 hours before the specimenprimary blood lymphocytes (PBLs). Patient or normal blood samples withheparin as an anti-coagulant were processed rapidly (within 2 hours ofbeing taken) and PBLs purified on histopaque. The PBLs were washedextensively (at least 3 times) to remove platelets and 10⁶ PBLs wereadded to each LN.increment.P dish in a total 4 ml DMEM+10% FBS. Thepositive control was 100 μl of a viral supernatant of the syntheticamphotropic murine retrovirus, MA, added to the LN.increment.Ps; thenegative control (apart from the normal individuals) was LN.increment.Pcells with no additions. On day seven, the secondary test line (eitherrat 208F cells or human LNSV cells), were plated at 1-2×10⁵ cells in 60mm dish in αMEM+10% FBS+polybrene. At least 4 hours later, thesupernatant was removed from the LN.increment.P/PBL dishes and filteredthrough 0.45μ filters or spun at 1500 rpm for 5 minutes in a clinicalcentrifuge to remove cells. 1-2 ml of clean supernatant was then addedto the secondary test line, plus 2 ml of αMEM+10% FCS+polybrene and thecells incubated 24 hours in 5% CO₂ at 37°. On day eight, the medium waschanged to αMEM+10% FBS plus HAT (10⁻⁴ M hypoxanthine, 2×10⁻⁷ MAminopterin, 5×10⁻⁵ M thymidine). The medium was subsequently changedevery 3 days and on day 18 the plates were washed, fixed, stained andcolonies counted.

Using the above protocol, a first fifty-two clinical specimens wereexamined, including forty-nine synovial fluids, two synovial biopsies,and one blood sample. Most of the forty-nine fluids were from patientswith RA, while the remainder included those with Reiter's Disease,osteoarthritis, gout, and a mono-articular chronic arthritis.Approximately two thirds of the RA specimens tested produced a positiveresult using the assay procedure described. One patient with Reiter'sDisease also tested positive, but had HTLV III in his cells, asdetermined by nucleic acid hybridization assay. Utilization of thedifferent cell lines described, as primary and secondary cells, did notsignificantly affect assay results. The titer of the retrovirus in thesynovial fluid specimens was relatively constant, in the range of 1 to30 plaques per milliliter.

Experiments also indicated that the activity in the samples from the RApatients appears to be cell-associated. In particular, plaque-formingactivity was observed more consistently in the material that issedimented by centrifugation, and not in the remaining supernatant.

Another experiment using Southern blot analysis demonstrated that thepLS.increment.P.increment.LM retroviral construct is actuallytransferred from the LN.increment.P line to the 208F cell line. Inparticular, the DNA was isolated from 208F cells from the above assayprocedure, which are HAT resistant, and a radioactive probe made fromthe pLS.increment.P.increment.LM vector construct detected acorresponding four kilobase (kb) fragment on that isolated DNA, cut withthe enzyme Sst I.

A further experiment used peripheral blood lymphocytes ("PBLs") isolatedby FICOLL HYPAQUE gradients from a patient with RA. It was shown, thatsuch lymphocytes can pseudotype the pLS.increment.P.increment.LM vectorconstructs from the LN.increment.P cells to the 208F cells. In a furtherexperiment, the primary cells consisted of U937 cells carrying aretroviral vector construct termed "Bag". Cepko, PNAS 84:156-160 (1987).The foregoing construct contains both β-galactosidase ("Bgal") gene andthe resistance gene for neomycin. A second set of U937 cells was used asthe secondary cells. The primary cells were then cultured with the samePBLs from the patient of the preceding example, using the assayprotocol. As shown below, the secondary cells were found to carry thebeta-galactosidase gene.

EXAMPLE 2

Primary blood lymphocytes were isolated on four different occasions fromone rheumatoid arthritis patient (WT), three other patients and 5 normalindividuals. These were cocultivated with LN.increment.P cells and afterthree (3) days the supernatants transferred to 208F cells which wereassayed for uptake of the HPRT vector by HAT selection. Column 1 ofTable 1 shows the supernatants from LN.increment.P cultivated withoutpatient or normal PBLs. Column 2 shows the patient or normal PBLscultivated with LN.increment.P. Column 3 shows patient or normal PBLplus 1 μl (patient) or 10 μl (normals) of MA viral supernatantcultivated with LN.increment.P. Column 4 shows one (1) or 10 μl of MAviral supernatant cocultivated with LN.increment.Ps. Results areexpressed as the number of clones seen after 10 days of HAT selectionand experiments were performed in triplicate. Unrelated 208F cells werealways selected in parallel and were always completely negative (notshown).

                  TABLE 1    ______________________________________           1.         2.        3.       4.    RA     LNΔP PBL       PBL + MA MA    Patients           Supernatant                      on LNΔP                                on LNΔP                                         LNΔP    ______________________________________    WT    Expt. #1           00,00,00   12,07,16  05,07,06 14,09,07    Expt. #2           00,00,00   06,01,02  04,00,06 03,10,07    Expt. #3           02,00,00   04,07,01  03,09,05 09,15,02    Expt. #4           00,00,00   05,03,06  06,05,10 10,10,05    CT     00,00,00   02,06,05  04,05,07 07,10,12    WR     00,00,00   03,04,03  08,12,05 10,12,09    JC     00,00,00   05,02,07  03,06,19 05,13,11    Normals    KT     00,00,00   00,00,00  04,--,-- 01,--,--    AG     00,00,00   00,00,00  68,82,63    ,100,92    BS     00,00,00   00,00,00  37,45,26 40,39,67    JD     00,00,00   00,00,00  17,06,-- 108,21,38    M      00,00,00   00,00,00  42,19,31 26,12,35    ______________________________________

EXAMPLE 3

The assay was carried out as in Example 2 except that the PBLs werecocultivated with LN.increment.P cells for up to 14 days and thesupernatants were transferred to 208F cells at 3 and 14 days. In thiscase, the assays at 14 days were performed without and with pretreatmentof 208F with polybrene (-polybrene, +polybrene) (TNTC=too numerous tocount). The results are shown in Table 2.

                  TABLE 2    ______________________________________                Time of cocultivation:                     14 days                3 days -polybrene +polybrene    ______________________________________    LNΔP (negative                  00,01,00 --         --    control)    MA (positive control)                  TNTC     TNTC       TNTC    RA Patients    1             00,01,00 28,09,01   13,00,--    2             00,00,00 01,02,01   03,01,00    3             00,04,00 01,03,02   01,16,20    4             01,00,00 02,01,01   03,00,01    5             00,00,00 00,02,00   00,03,05    ______________________________________

EXAMPLE 4

Primary Blood Lymphocytes from an RA patient (WT) were isolated onFICOLL HYPAQUE gradients and 5×10⁶ cells cultivated in RPM1 1640 plus10% FCS+PHA with 10⁴ cfu of Bag vector (Price et al., Proceedings of theNational Academy of Sciences, 84:156-160 (1987)), that had been shown tobe helper free (<1 unit of competent virus/ml). After 3 days, 1 ml ofthe supernatant was transferred into U937 cells growing in the samemedium and 24 hours later the cells were fixed and stained for betagalactosidase activity (Price et al., supra), and blue (positive) cellscounted to test for transfer of the Bgal-containing vector from the PBLto the U937 cells (Experiment #1). Controls were Experiment #2: 10⁸units MA virus added to U937 cells, staining after 24 hours; Experiment#3: 10⁴ units Bag vector added to U937 cells, staining after 24 hours;and Experiment # 4: 10⁸ units MA virus, 10⁴ units Bag added to patientscells, cocultivated for 3 days, the supernatant transferred to U937cells, stained after 24 hours. Previous experiments showed that 10⁴units of Bag vector incubated in medium at 37° C. for 3 days had nodetectable infective activity on U937 cells. The results are shown inTable 3.

                  TABLE 3    ______________________________________    Experiment             Blue cells/10.sup.5 U937 cells    ______________________________________    #1    (Bag + PBL)        6        10          (onto U937)    #2    (U937 + Bag)     >10.sup.3  >10.sup.3    #3    (U937 + MA)        0         0    #4    (Bag + PBL + MA) >100       --          (onto U937)    ______________________________________

Thus, the appearance of blue cells, even at a low level can be used toassay for the presence of a retrovirus in this quick (4 days) assay.

The complementation assay as used in the foregoing Examples relies on anunknown retrovirus providing complementing viral proteins to package aretroviral vector based on a murine leukemia virus (MLV) backbone. Thisvector has then to exit the primary cell and enter a second cell linewhere the presence of the vector is assayed. The packaging, exit andinfection by the vector depend on the unknown viral proteins beingcapable of recognizing and packaging, albeit at low efficiency, a vectorwhich may be at a large evolutionary distance from it. In addition, theenvelope proteins of the unknown virus may allow only low efficiencyentry into the secondary tester cell line. To increase the chances thatthe retrovirus being tested for will successfully complement thereplication defective retroviral vector carrying the marker sequence, aminimum complementation assay was designed, a particular example ofwhich is provided in Example 5 below.

EXAMPLE 5

In this particular example of a minimum complementation assay,complementing defective viral genomes were designed which express allbut one Moloney MLV viral protein function. These are transfected intothe primary tester cell line carrying the Moloney MLV vector. Thus theunknown retrovirus has only to supply one function (i.e., minimalcomplementation) in trans to allow production of marker vector plasmid,such as the HPRT vector in LN.increment.P. It is important in thiscontext that LN.increment.P is not a mouse cell line, since such linesare capable of repairing such defective MLV genomes. The overall effectis to make the assay:

(1) more sensitive to a particular type of unknown retrovirus;

(2) capable of detecting a wider range of retroviral types.

A number of separate in frame deletions were made in the indicateddomains of the cloned Moloney MLV genome pMLV-K (see Table 4 below andFIG. 1), by deletion of restriction enzyme fragments or by site directedoligonucleotide mutagenesis to produce 8 different genomes each with theindicated single deletion. The nucleotide numbers after the restrictionsites correspond to the numbering for Moloney MLV in "RNA TumorViruses", Vol. 2, 1985, Cold Spring Harbor Laboratory. Preparation ofdeletion mutants in Table 4 below are prepared according to well knowntechniques. For example, see T. Maniatis, "Molecular Cloning--ALaboratory Manual", Cold Spring Harbor Laboratory, N.Y., U.S.A. (1982),and T. Kunkel, PNAS, Vol. 82, p. 488 (1985).

                  TABLE 4    ______________________________________    DELETION MUTANTS OF Moloney MLV                                         Amino                                Exact    Acid                       Deletion Deletion Dele-    Deletion Size      Domain   bR       tion    ______________________________________    1)  p700    Hind III(4894)/Sph I                               Integrase                                      243     81                (5137)    2)  p800    Sal I(3705)/Hind III                               RT     1185   395                (4894)    3)  p900    Hpa I(5816)/Hpa I                               Env    1380   460                (7195)    4)  p1100   Hind III(4894)/Sac II                               Integrase                                       54     18                (4949)    5)  p1200   By mutagenesis Protease                                      201     67    6)  p1600   Bal I(1672)/Bal I                               P30    381    127                (2053)    7)  p1400   Bgl II(1906)/Bst E2                               P30,10,14                                      543    181                (2453)    8)  p1700   Sta I(2689)/Sta I                               RT     933    311                (3622)    ______________________________________

An assay can then be performed using any of these modifiedLN.increment.P lines as before in the preceding Examples("complementation assay"). Likewise, corresponding viral vectors couldbe carried in retroviruses and used in an assay to determine thepresence or amount of a retrovirus in specimen cells. The complementarydefective MLV and viral vector can also be provided in other suitablehuman or other species cell types. In addition, the secondary testerused in the preferred embodiment of the original assay is susceptible toinfection with ecotropic MLV (such as pMLV-K). Moloney MLV is availablefrom American Type Culture Collection (Rockville, Md., U.S.A.) underdeposit number ATCC VR-190, the MLV-K strain can be prepared from MLV asdescribed by Miller et al. Molec. Cell. Biol., Vol. 5, p. 431-437(1985). Thus the ecotropic envelope will "help" in this case. If other,for example, human lines are used as secondary tester lines, the primarytester line will have (in addition to the vector and complementarydefective viral genome) a genome encoding an envelope capable of"helping" infection of human cells, such as the amphotropic envelope.(see FIG. 2).

Various modifications and alterations to the embodiments to theinvention described above, can be envisaged by one skilled in the art.Accordingly, the present invention is not limited to the specificembodiments described above.

We claim:
 1. A method of testing for the presence or amount of aretrovirus in a specimen, comprising the following steps in order:(a)exposing a replication defective retroviral vector having a markersequence and carried by primary cells to said specimen, in the absenceof any other added virus which would allow said replication defectivevector to produce a recombinant retrovirus by coinfecting said primarycells, such that a retrovirus which may be infecting said specimen cancomplement the replication defective retroviral vector and produce arecombinant retrovirus carrying the marker sequence; (b) culturing saidprimary cells with the specimen; (c) testing for the presence of arecombinant retrovirus; and thereby (d) determining whether saidretrovirus is present in said specimen, wherein a positive result instep (c) indicates that said retrovirus is present in said specimen. 2.A method as defined in claim 1 wherein the replication defectiveretroviral vector is in proviral form.
 3. The method of claim 1, whereinthe replication defective retroviral vector carries a sequence codingfor a packaging protein.
 4. The method of claim 3, wherein thereplication defective retroviral vector carries sequences coding for allthe packaging proteins necessary for replication except for one missingpackaging protein, so that the retrovirus being tested for need onlyproduce the missing protein in order to produce the recombinantretrovirus carrying the marker sequence.
 5. The method of claim 1,wherein said specimen comprises a group of cells, said methodadditionally comprising the step of dividing said specimen intoaliquots, each of said aliquots comprising one or more cells from saidgroup of cells, wherein each of a plurality of groups of replicationdefective retroviral vectors is exposed to only one of said aliquots ofsaid specimen, the retrovital vectors of each group having a markersequence and a sequence which codes for a packaging protein that isdifferent from the packaging proteins coded for by the sequences of theremaining groups, and wherein the sequences which code for all thepackaging proteins required to produce a recombinant retrovirus carryingsaid marker sequence are present in the sequences of said groups ofvectors.
 6. The method of claim 1, wherein the defective retroviralvector has at least one cis-acting sequence corresponding to that of aretrovirus of a type different from the retrovirus being test for. 7.The method of claim 1, wherein said specimen comprises tissue from amammal.
 8. A method as defined in claim 1 further comprising the step ofwashing the primary cells and further culturing the primary cells withthe specimen prior to culturing the secondary cells with the primarycell medium so as to reduce the concentration of any inactiverecombinant retrovirus which may otherwise be present in the primarycell medium.
 9. The method of claim 1, wherein said retrovirus infectingsaid specimen is an unknown retrovirus.
 10. A method of testing for thepresence or amount of a retrovirus infecting a specimen, comprising thefollowing steps in order:(a) culturing said specimen with primary cellsdisposed in a primary cell medium which carry a replication defectiveretroviral vector having a marker sequence, in the absence of anotheradded virus which would allow said replication defective vector toproduce a recombinant retrovirus by coinfecting said primary cells, suchthat a retrovirus, if present, may complement the replication defectiveretroviral vector and produce a recombinant retrovirus carrying themarker sequence; (b) culturing secondary cells with said primary cellmedium so that said recombinant retrovirus carrying the marker sequence,if present, infects said secondary cells; (c) testing for the presenceor amount of the marker sequence in the secondary cells; and thereby (d)determining whether said retrovirus is present in said specimen, whereina positive result in step (c) indicates that said retrovirus is presentin said specimen.
 11. A method as defined in claim 10 wherein thereplication defective vector is a vector construct.
 12. The method ofclaim 10, wherein said marker sequence is a nucleic acid sequenceencoding a protein selected from the group consisting of an enzyme, aprotein which will enable growth of the secondary cells in a medium inwhich said cells would not otherwise grow, and an antigen.
 13. Themethod of claim 10, wherein the defective retroviral vector has acis-acting sequence corresponding to that of a retrovirus of a typedifferent from the retrovirus being tested for.
 14. The method of claim10, wherein the replication defective retroviral vector carries asequence which can produce at least one packaging protein in the primarycells.
 15. The method of claim 10, wherein the replication defectiveretroviral vector carries sequences coding for all the packagingproteins necessary for replication except one missing packaging protein,so that the retrovirus being tested for need only produce the missingprotein in the primary cells to produce the recombinant retrovirus carrythe marker sequence.
 16. The method of claim 10, wherein the secondarycells are from different cell lines than the primary cells.
 17. A methodas defined in claim 10 wherein the retroviral vector constructs carriedby some of the primary cells contain a different packaging signal fromthe retroviral vector constructs carried by others of the primary cells.18. A method as defined in claim 10 additionally comprising treating atleast one of the primary and secondary cells with a detergent to enhancethe ability of the cells to be infected with a recombinant retrovirus.19. The method of claim 10, wherein said retrovirus infecting saidspecimen is an unknown retrovirus.
 20. The method of claims 1 or 10,wherein the primary cells used are from a plurality of different celllines.
 21. The method of claims 1 or 10, wherein the retrovirus beingtested for is rheumatoid arthritis associated retrovirus.